RNA interference (RNAi) is an evolutionarily conserved, sequence-specific mechanism triggered by double-stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus et al., Nature Rev. Genet., 3:737 (2002)). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active “short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides in length (Elbashir et al., Genes Dev., 15:188 (2001)). siRNA can be used to downregulate or silence the transcription and translation of a gene product of interest, i.e., a target sequence.
As part of the innate defense mechanism against invading pathogens, the mammalian immune system is activated by a number of exogenous RNA (Alexopoulou et al., Nature, 413:732-738 (2001); Heil et al., Science, 303:1526-1529 (2004); Diebold et al., Science, 303:1529-1531 (2004)) and DNA species (Krieg, Ann. Rev. Immunol., 20:709-760 (2002)), resulting in the release of interferons and inflammatory cytokines. The consequences of activating this response can be severe, with local and systemic inflammatory reactions potentially leading to toxic shock-like syndromes. These immunotoxicities can be triggered by very low doses of an immunostimulatory agent, particularly in more sensitive species, including humans (Michie et al., N. Engl. J. Med., 318:1481-1486 (1988); Krown et al., Semin. Oncol., 13:207-217 (1986)). It has recently been demonstrated that synthetic siRNA can be a potent activator of the innate immune response when administered with vehicles that facilitate intracellular delivery (Judge et al., Nat. Biotechnol., 23:457-462 (2005); Hornung et al., Nat. Med., 11:263-270 (2005); Sioud, J. Mol. Biol., 348:1079-1090 (2005)). Although still poorly defined, immune recognition of siRNA is sequence dependent and likely activates innate immune cells through the Toll-like receptor-7 (TLR7) pathway, causing potent induction of interferon-alpha (IFN-α) and inflammatory cytokines. Toxicities associated with the administration of siRNA in vivo have been attributed to such a response (Morrissey et al., Nat. Biotechnol., 23:1002-1007 (2005); Judge et al., supra).
Stabilization of synthetic siRNA against rapid nuclease degradation is generally regarded as a prerequisite for in vivo and therapeutic applications. This can be achieved using a variety of stabilization chemistries previously developed for other nucleic acid drugs, such as ribozymes and antisense molecules (Manoharan, Curr. Opin. Chem. Biol., 8:570-579 (2004)). These include chemical modifications to the native 2′-OH group in the ribose sugar backbone, such as 2′-O-methyl (2′OMe) and 2′-Fluoro (2° F.) substitutions that can be readily introduced into siRNA as 2′-modified nucleotides during RNA synthesis. Although a number of reports have demonstrated that chemically stabilized siRNA containing 2′OMe (Czauderna et al., Nucl. Acids Res., 31:2705-2716 (2003); Allerson et al., J. Med. Chem., 48:901-904 (2005); Prakash et al., J. Med. Chem., 48:4247-4253 (2005)), 2° F. (Chiu et al., RNA, 9:1034-1048 (2003); Layzer et al., RNA, 10:766-771 (2004); Allerson et al., supra; Prakash et al., supra), 2′-deoxy (Chiu et al., supra), or “locked nucleic acid” (LNA) (Hornung et al., supra; Elmen et al., Nucl. Acids Res., 33:439-447 (2005)) modifications can be designed that retain functional RNAi activity, such modifications appear to be tolerated only in certain ill-defined positional or sequence-related contexts. In fact, the introduction of chemical modifications to native siRNA duplexes can, in many cases, have a negative impact on RNAi activity (Hornung et al., supra; Czauderna et al., supra; Prakash et al., supra; Chiu et al., supra; Elmen et al., supra). As a result, the design of chemically modified siRNA has required a stochastic screening approach to identify duplexes that retain potent gene silencing activity.
Poor uptake of exogenous nucleic acids by cells represents an additional barrier to the development of siRNA-based drugs. siRNA can be encapsulated within liposomes termed stable nucleic acid-lipid particles (SNALP), which enhance intracellular uptake of nucleic acids and are suitable for systemic administration. These systems are effective at mediating RNAi in vitro (Judge et al., supra) and have been shown to inhibit viral replication at therapeutically viable siRNA doses in a murine model of hepatitis B (Morrissey et al., supra). However, these studies were performed with synthetic siRNA that included greater than 90% modified nucleotides, which may compromise the potency of RNAi-mediated gene silencing.
Thus, there is a strong need in the art for minimally modified siRNA molecules that abrogate the immunostimulatory activity of siRNA without having a negative impact on RNAi activity. The present invention addresses this and other needs.